The present invention relates to screening complex biological materials, such as natural samples, natural products, and combinatorial libraries, for active compounds such as affinity ligands. In particular, active compounds are extracted using size-exclusion separation and ultrafiltration, and are subjected to subsequent analysis. The analysis may include structural or functional characterization of an isolated active compound by any one of many techniques known to one of ordinary skill in the art of screening complex biological materials. Suitable analytical techniques include, but are not limited to, mass spectrometry, liquid and other chromatography, a secondary assay, and the like.
Testing complex biological samples for new drug candidates in high throughput screening programs is a successful strategy employed by the pharmaceutical industry. Once an active sample is identified, however, it can be difficult to isolate the active compound, particularly from natural product extracts or natural samples.
Natural samples such as natural extracts represent a highly and chemically diverse collection of compounds that include very small to very large molecules, which makes it very difficult to isolate any single active compound. An active compound is one that has an effect on a biological molecule such as a protein or nucleic acid. For instance, active compounds include affinity ligands that can bind to the biological molecule. As most successful drug compounds are of small molecular weight (less than 2,000 Daltons), separations based on size can be a useful tool in assisting the isolation of active components from natural samples. The present invention enables high-throughput screening of such complex biological materials for identification of active ligands to a biological target molecule.
Combinatorial chemistry offers the means to generate a large number of different chemicals simultaneously. Modern analytical methods allow the screening of such combinatorial libraries to select those compounds possessing desirable properties. However, these methods have their limitations, so that a need remains, too, for a successful analytical methodology that provides high throughput screening of combinatorial libraries against biological targets for identification of active ligands.
Screening a library generally involves a binding or a functional assay to determine the extent of ligand-receptor interaction. Often, either the ligand or the receptor is immobilized on a solid surface (e.g. polymer bead or plate) and, after detection of the binding or the functional activity, the ligand is released and identified by a different means, for example, by mass spectrometry. Solid-phase screening assays offer faster isolation and identification of active analytes compared to the solution-based methods. On the other hand, limitations associated with heterogeneous assays create a demand for a breakthrough technology for rapid and efficient screening of natural samples screened in solution. Solution-phase assays are desirable to increase screening specificity, but current methodologies involve iterative processes that are long and laborious.
Recently, electrospray ionization mass spectrometry (ESI-MS) has also been used in screening, directly or in conjunction with a solution-based screening method. The direct screening of combinatorial libraries by ESI-MS relies on its ability to characterize non-covalent complexes of proteins bound to ligands (i.e., to distinguish between a protein target and its ligand, even a small one). Alternatively, mass spectrometry can be coupled, on-line or off-line, with solution-based screening methodologies, as a final dimension for structure determination of biologically active compounds.
One method, the Hummel-Dreyer method, recently used for binding studies of small molecules to proteins, is based on size separation of the receptor protein and its ligand by gel filtration. The approach of separating a receptor (i.e., a target protein) and a receptor-ligand complex from other small, unbound molecules on the basis of size differences, was first applied to the separation of antibody-peptide complexes from the rest of a peptide library, with subsequent analysis of the ligands on reversed-phase high-performance liquid chromatography (RP-HPLC) (R. N. Zuckerman et al., Proc. Nat. Acad. Sci., USA 89:4505-4509 (1992), J. M. Kerr et al., Bioorg. Med. Chem. Lett. 3:463=468 (1993)).
Size-exclusion separation has also been applied to small molecule combinatorial libraries. Some work has been done on developing a methodology for selection and identification of ligands with high affinity to a biological target using a size-exclusion-complex isolation procedure and mass spectrometry: e.g., Y. Dunayevskiy et al., Rapid Comm. Mass Spec., 11:1178-84 (1997); M. M. Siegel et al., J. Mass Spec., 33:264-273 (1998). These attempts to screen combinatorial libraries and other complex mixtures of compounds on the basis of size-exclusion separation have had limited success in actual application, particularly under the rigors of high throughput screening. One major problem has been the tendency of transfer lines and other conduits used in high-throughput, size-exclusion-complex isolation screening protocols, particularly transfer lines to reverse phase HPLC column, to become clogged and impassable after only 1-2 hours of operation. Such clogging, which occurs due to irreversible collection of the protein on the stationary phase of the HPLC column, necessitates frequent changes of the transfer lines. It also is a limiting factor in the overall effectiveness and on-line automation of such screening protocols. This effect is even more pronounced when dealing with complex biological mixtures such as natural samples that contain large MW biomolecules. Therefore, a need has remained for a size-exclusion-based screening method that can effectively extract active ligands from natural samples and simultaneously withstand high throughput conditions.